An estimated 1 million Americans suffer from chronic inflammatory bowel disease (IBD) and 20 million Americans suffer from irritable bowel syndrome (IBS). IBD, comprised of both Crohn's Disease (CD) and ulcerative colitis (UC), is characterized by a chronic inflammatory response that results in histologic damage to the intestinal lining. Both CD and UC exhibit large numbers of leukocytes that migrate to the mucosa and into the intestinal lumen. Both diseases oscillate between active (i.e., presence of intestinal inflammation) and inactive (i.e., minimal to no intestinal inflammation) stages of disease activity. Active IBD can include symptoms such as bloody diarrhea, abdominal pain, and fever. The inactive stage has minimal to no intestinal inflammation and lacks severe gastrointestinal illness.
Patients who have active IBD but who exhibit mild signs and symptoms may be difficult to distinguish from patients with active IBS, an intestinal disorder of motility and the intestinal nervous system. Unlike IBD, IBS does not involve intestinal inflammation. In persons with IBS, the intestine appears normal upon endoscopic examination and leukocytes are not present in the mucosa or in fecal specimens. Symptoms can mimic those of IBD and include bloating, diarrhea, constipation, and severe and often debilitating abdominal pain. It is estimated that at least 20 million Americans suffer from IBS.
The similarity in symptoms between IBS and IBD renders rapid diagnosis difficult. However, given the potential severity of untreated IBD, differential diagnosis is crucial. The diagnosis of gastrointestinal illnesses, in general, is aided by diagnostic tests such as enzyme-linked immunosorbant assays (ELISAs), latex agglutination and lateral flow immunoassay. These tests are rapid and inexpensive methods for detecting markers in feces for enteric pathogens and inflammation. One marker of particular interest that has been found to be most specific for leukocytes in fecal specimens is lactoferrin. Human lactoferrin is an 80 kilodalton glycoprotein. This iron-binding protein is secreted by most mucosal membranes. It is a major component of the secondary granules found in polymorphonuclear neutrophils (PMNs), a primary component of the acute inflammatory response. Other hematopoietic cells such as monocytes and lymphocytes, do not contain lactoferrin, whereas various bodily secretions contain levels in the mg/mL range. During the process of inflammation, PMNs infiltrate the mucosa lining of the small and large intestine. This increase in the number of activated tissue leukocytes and exudation of plasma from ulcerated mucosa results in an increase in the level of lactoferrin found in feces. The protein is resistant to proteolysis and, as such, it provides a useful non-invasive fecal marker of intestinal inflammation.
Human lactoferrin has been used as a marker for fecal leukocytes in a number of applications. For instance, fecal lactoferrin has been used as a marker for leukocytes to distinguish noninflammatory diarrhea from inflammatory diarrhea, as disclosed in U.S. Pat. No. 5,124,252. Noninflammatory diarrhea caused by agents such as rotavirus, Norwalk-like agents and cholera, typically causes minimal to no intestinal damage and patients respond readily to oral rehydration. Inflammatory diarrheas include those caused by enteric pathogens such as Clostridium difficile, Shigella species, Salmonella species, Campylobacter jejuni and Entamoeba histolytica and those that have no clearly defined infectious agent such as CD and UC. U.S. Pat. No. 5,124,252 discloses an in vitro test for fecal leukocytes that aids in distinguishing inflammatory from noninflammatory diarrhea. The '252 patent discloses testing fecal samples suspected of containing leukocytes with an assay that utilizes an antibody for lactoferrin to determine the presence of leukocytes in the fecal sample.
Human lactoferrin also has been used as a marker for diagnosis of inflammatory gastrointestinal disorders, colon polyp and colorectal cancer as disclosed in U.S. Pat. No. 5,552,292. However, neither the method of the '252 patent nor that of the '292 patent disclose utility in distinguishing IBS from IBD. The samples tested by the assay of the '252 patent are samples suspected of containing leukocytes. This suspicion is owed to the patient presenting with diarrhea. However, 25-50% of persons having IBD do not present with diarrhea and, thus, the '252 patent does not relate to diagnosing etiology in such patients. As for the '292 patent, the disclosed method utilizes a 1:100 sample dilution which does not allow for accurate quantitation of lactoferrin levels. Further, the '292 patent discloses using partial forms of molecules for testing and not total endogenous lactoferrin, again affecting the accuracy of the quantitation. The method of the '292 patent also does not relate to utilizing lactoferrin levels to distinguish IBD from IBS. The population tested in the '292 patent, while including persons with UC and CD, did not include persons having IBS.
IBD is comprised of both Crohn's disease and ulcerative colitis. These two distinct diseases require a rapid differential diagnosis for optimal treatment. Crohn's disease may involve the entire gastrointestinal tract and include inflammation extending into the transmural mucosa, whereas ulcerative colitis affects solely the large bowel and includes inflammation of the innermost lining. Conventional methods to differentiate between Crohn's disease and ulcerative colitis utilizing multiple endoscopy examinations and histological analysis may take years to confirm a diagnosis.
U.S. Pat. No. 6,218,120 discloses a method of determining the presence of serum ANCA as a marker to diagnose IBD. However, it does not disclose a method for diagnosing ulcerative colitis in a patient diagnosed with IBD.
Serological methods for the differential diagnosis of CD and UC also are known in the art. For example, it is known to use the presence of serum anti-Saccharomyces cerevisiae antibodies (ASCA) to diagnose CD. See Main et al., Antibody to Saccharomyces cerevisiae (baker's yeast) in Crohn's disease, BMJ Vol. 297 (Oct. 29, 1988); Broker et al., A Murine Monoclonal Antibody Directed Against a Yeast Cell Wall Glycoprotein Antigen of the Yeast Genus Saccharomyces, FEMS Microbiology Letters 118 (1994), 297-304. It is further known in the art to use the presence of serum ASCA to diagnose clinical subtypes of UC and CD in patients presenting with established diagnoses. For example, U.S. Pat. No. 5,968,741 discloses utilizing the presence of serum ASCA to diagnose a medically resistant clinical subtype of UC in patients presenting with an established diagnosis of UC. Similarly, U.S. Pat. No. 5,932,429 discloses utilizing the presence of serum ASCA to diagnose a clinical subtype of CD in patients presenting with an established diagnosis of CD.